It is the long-term objective of this study to understand the genetic control of root development and periodontium formation. Advances in molecular and developmental biology have linked the function of numerous genes to specific stages of organ development. In the case of tooth development, more than 300 hundred genes have been reported as having some role in this process. As expected, mutations in these genes can result in dental defects which vary in severity depending on how the mutation affects the function of that protein or what process that particular gene regulates. Much of the knowledge relating a gene to its function is derived from the use of transgenic animals which has allowed the creation of animal models for genetic diseases. In the case of tooth formation, many of the genes involved in the early stages of tooth development and crown formation have been established. A few years ago we reported that the loss of function of the transcription factor Nuclear Factor I-C (NFI-C) in mice results in a unique phenotype characterized by molars with very short or no roots at all. No other phenotype was found and no defects in the crowns were detected indicating that the NFI-C gene product is essential for the process of root formation. NFI- C is the first and up to date only gene that has been found to regulate root development and therefore periodontium formation. Root formation, like crown formation, is dependent on epithelial-mesenchymal interactions resulting in differentiation of the corresponding tissues and production of the mineralized extracellular matrices. Since the main difference between the crown and root is its epithelial component which in the crown is composed by the outer and inner enamel epithelia separated by the stellate reticulum and the stratum intermedium. The root contains only inner and outer enamel epithelia forming the structure known as Hertwig's Epithelial Root Sheath (HERS). Based on this difference and data presented in the preliminary data section, we propose to test the central hypothesis that Nfi-c controls periodontium development by regulating the function of HERS which in turn control root morphogenesis, odontoblast differentiation, dentin and cementum production and periodontal ligament formation. We propose to test this hypothesis by realizing the following Specific Aims: establish the role of NFI-C on HERS differentiation and maturation;determine NFI-C down-stream genes associated with HERS differentiation and maturation;determine NFI-C interactions in cells associated with root development;determine the mechanism by which HERS control odontoblast differentiation and determine the mechanism by which HERS control periodontal ligament and cementum formation. These studies will use the NFI-C knockout mice as well as our established immortal cell lines for the tissues associated with root formation, shRNA and microarray technology. The data generated in this study will provide much needed information related to the process of normal and abnormal root and periodontium formation and the regulatory factors involved in this process. This data is of great health relevance to understand genetic mutations resulting in root defects, root agenesis and/or premature tooth loss which is critical to establish future genetical and tissue engineering therapies for periodontium regeneration. PUBLIC HEALTH RELEVANCE: It is the long-term objective of this study to understand the process of root development and periodontium formation at the morphological, cellular and molecular levels. This includes the mechanisms underlying root dentin, cementum and periodontal ligament formation which lead to root attachment in the healthy periodontium. This application will focus on the role of the transcription factor NFI-C which appears to be essential for periodontium formation. The data generated in this study is of great health relevance to understand genetic defects resulting in root agenesis and periodontium defects which result in premature tooth loss. Additionally, understanding root and periodontium development is critical to establish genetical and tissue engineering therapies for periodontium regeneration.